2.8.1. LDH-Glo Cytotoxicity Assay

A bioluminescent lactate dehydrogenase (LDH)-release assay (LDH-Glo Cytotoxicity Assay, Promega) was used to assess cytotoxicity in supernatants of cells treated with liposomes according to manufacturer’s instruction. Briefly, 2 µL of medium was collected from each well at the end of the treatment, and diluted in the LDH storage buffer (200 mm Tris-HCl (pH 7.3, 10% glycerol, 1% BSA). The LDH storage buffer + medium solution was subsequently mixed in a 1:1 ratio with LDH detection reagent in a white 96-multiwell plate (Corning, NY, USA), and incubated for 30 min at room temperature. Luminescence was measured using the Mithras LB 940 instrument (Berthold technologies, Bad Wildbad, Germany), with an integration time of 0.8 s. Maximum LDH Release Control was obtained lysing untreated cells with 10% Triton X-100 (2 μL, Sigma-Aldrich, Milan, Italy). In each experiment, we calculated the % of cytotoxicity as follows:

where MB is Medium Background.